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X chromosome inactivation in the mouseNorris, Dominic Paul January 1995 (has links)
No description available.
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Investigating the contribution of the non-coding gene Ftx to X-chromosome inactivation in mammals / Étude de la contribution du gène non-codant Ftx dans l'inactivation du chromosome X chez les mammifèresFurlan, Giulia 23 September 2016 (has links)
L’inactivation du chromosome X (XCI) est un mécanisme qui permet l’extinction transcriptionelle d’un des deux chromosomes X chez la femelle. XCI est régulé par une région spécifique nommée centre de l’inactivation du chromosome (Xic), contenant plusieurs gènes produisant de longs ARNs non codants (lncRNAs). Parmi ces lncRNAs, le transcrit Xist est l’effecteur principal pour l’XCI. Xist peut s’accumuler en cis sur le chromosome et recruter la machinerie qui permettra l’initiation et la propagation de l’extinction transcriptionnelle à l’échelle du chromosome.Le laboratoire d’accueil a identifié un nouveau gène du Xic qui produit le lncRNA Ftx. Dans cette étude, on a pu montrer que l’inactivation du chromosome X est fortement perturbée dans les cellules Ftx-/- et s’accompagne par une forte baisse du niveau d’expression et d’accumulation de Xist. Dans ce contexte, certaines cellules parviennent à maintenir l’expression de Xist mais le profil de couverture du chromosome X par Xist est anormal, présentant un profil diffus ; ceci est associé à une extinction transcriptionnelle déficiente des gènes liés à l’X. Dans les lignées hétérozygotes Ftx+/-, l’expression et l’accumulation de Xist est aussi affectée mais dans une moindre mesure, si bien qu’il apparaît que le nombre de copies de Ftx soit important pour sa fonction. Par ailleurs, l’inactivation du chromosome X dans les cellules Ftx+/- est biaisée de telle sorte que le chromosome X portant une copie fonctionnelle de Ftx est préférentiellement inactivé, suggérant un rôle en cis de Ftx. Ces résultats montrent que Ftx est un activateur de Xist et qu’il est essentiel pour la mise en place de l’inactivation / X-chromosome inactivation (XCI) is a female-specific, chromosome-wide regulatory process that, in eutherians, ensures dosage compensation for X-linked genes between sexes. XCI is controlled by a cis-acting locus on the X-chromosome, the X-inactivation center (Xic), enriched in genes producing long non-coding RNAs (lncRNAs). The Xic-linked gene Xist is the master player of XCI, and produces a lncRNA that accumulates in cis on the X-chromosome and recruits the machinery responsible for initiation and propagation of silencing.The laboratory has identified an additional Xic-linked non-coding gene, Ftx. In this study, we could find that, in female Ftx-/- lines, XCI is strongly impaired, with a significant decrease in the levels of Xist expression and in the percentage of cells showing normal Xist accumulation patterns. Importantly, a high proportion of the cells that still retain Xist expression show abnormal X-chromosome coating and a decreased ability to silence X-linked genes. These data reveal that Ftx is a positive Xist regulator and it is required for proper XCI establishment. In female Ftx+/- lines, the levels of Xist expression and the percentage of cells showing normal Xist accumulation patterns are also decreased, albeit to a lower extent compared to Ftx-/- lines, suggesting that Ftx works in a copy-dependent manner. In addition, a high proportion of Ftx+/- cells display skewed X-inactivation, with preferential inactivation of the wild-type X chromosome. This suggests that Ftx role on Xist accumulation is mostly restricted in cis. Taken together, these results demonstrate that Ftx is required for XCI establishment, where it functions as a strong Xist activator
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Investigation of the X inactivation centre region in miceHorn, Jacqueline Morag January 2000 (has links)
No description available.
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Análise funcional dos genes Xist e DNMT1 na manutenção do processo de inativação do cromossomo X humano através do silenciamento gênico por RNAi / Functional analysis of XIST and DNMT1 genes in the maintenance of X chromosome inactivation process in human through gene silencing by RNAiStabellini, Raquel 27 June 2008 (has links)
A inativação do cromossomo X (ICX) é o fenômeno através do qual um dos cromossomos X das fêmeas de mamíferos é silenciado para atingir compensação de dose em relação aos machos. Ela envolve a expressão do gene XIST exclusivamente no X inativo, e a associação em cis de seu RNA nesse cromossomo. Isso inicia a imposição de várias marcas epigenéticas no cromossomo X inativo, que garantem a manutenção deste estado de silenciamento transcricional de maneira estável durante todas as mitoses num organismo. Uma dessas modificações epigenéticas é a metilação do DNA, desempenhada principalmente pela enzima DNMT1. Os papéis de XIST e DNMT1 na manutenção da inativação do cromossomo X ainda são controversos em humanos, e nesse sentido foi objetivo desse trabalho analisar a possível função desses genes nesse processo em células humanas não transformadas. Foi otimizado um sistema experimental para o estudo de possíveis perturbações na manutenção da inativação do cromossomo X, onde a re-expressão de genes submetidos a esse processo pode ser monitorada. Nesse sistema foram identificados dois genes, MAOA e GYG2, cujo padrão de expressão no X inativo difere do previamente descrito. Demonstrou-se que baixos níveis de expressão do gene XIST foram suficientes para manter seu RNA associado ao X inativo, conservando o estado silenciado desse cromossomo. Além disso, foram obtidos indicativos de que a inibição de XIST em fibroblastos humanos gera uma diminuição da viabilidade celular. Foi possível demonstrar que DNMT1 é necessária para a manutenção da metilação global do genoma em células humanas não transformadas, e que eXISTe um mecanismo de compensação da inibição desse gene que leva ao aumento da expressão de DNMT3B. Ainda se observou que a repressão de DNMT1 não é suficiente para levar à reativação de genes no cromossomo X inativo. Além disso, a desmetilação encontrada nos promotores de MAOA e XIST não foi suficiente para levar à expressão destes genes nos cromossomo X inativo e ativo, respectivamente. Estes resultados enfatizam a necessidade de se estudar os mecanismos moleculares da ICX em humanos utilizando sistemas experimentais adequados para a análise de herança epigenética. / X chromosome inactivation (XCI) is the phenomenon through which one of the X chromosomes in female mammals is silenced to achieve dosage compensation related to males. It involves the expression of XIST gene exclusively from the inactive X, and the association of its RNA in cis in this chromosome. This leads to a series of epigenetic modifications in the chromatin of the inactive X (Xi) that guarantee a stable maintenance of the transcriptional silence through all the mitoses in the organism. One of these epigenetic modifications is DNA methylation, achieved mainly by the maintenance DNA methylase DNMT1. The roles of XIST and DNMT1 in the maintenance phase of XCI are controversial in humans. Therefore, the main goal of this present work was to analyze some of the possible functions of these genes in this process in untransformed human cells. An experimental system was optimized to study possible disturbances in maintenance of XCI, where the re-expression of genes submitted to this process could be monitored. In this system we identified two genes, MAOA and GYG2, whose pattern of expression on the Xi, differed from what had been previously described. It was demonstrated that low levels of XIST expression were sufficient to keep its RNA associated to the Xi, assuring the silenced state of this chromosome. Besides, evidences have been found that XIST inhibition in human fibroblasts reduces cellular viability. It was possible to demonstrate that DNMT1 is necessary to the maintenance of global genome methylation in untransformed human cells, and the eXISTence of a compensation mechanism involving DNMT3B upregulation. It was also observed that repression of DNMT1 was not sufficient to reactivate genes of the Xi chromosome. Additionally, demethylation of MAOA and XIST promoters was not enough to cause expression of these genes on the inactive and active Xs, respectively. All these results emphasize the requirement of studying the molecular mechanisms of XCI in humans using experimental systems appropriate for the analysis of epigenetic inheritance.
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Análise funcional dos genes Xist e DNMT1 na manutenção do processo de inativação do cromossomo X humano através do silenciamento gênico por RNAi / Functional analysis of XIST and DNMT1 genes in the maintenance of X chromosome inactivation process in human through gene silencing by RNAiRaquel Stabellini 27 June 2008 (has links)
A inativação do cromossomo X (ICX) é o fenômeno através do qual um dos cromossomos X das fêmeas de mamíferos é silenciado para atingir compensação de dose em relação aos machos. Ela envolve a expressão do gene XIST exclusivamente no X inativo, e a associação em cis de seu RNA nesse cromossomo. Isso inicia a imposição de várias marcas epigenéticas no cromossomo X inativo, que garantem a manutenção deste estado de silenciamento transcricional de maneira estável durante todas as mitoses num organismo. Uma dessas modificações epigenéticas é a metilação do DNA, desempenhada principalmente pela enzima DNMT1. Os papéis de XIST e DNMT1 na manutenção da inativação do cromossomo X ainda são controversos em humanos, e nesse sentido foi objetivo desse trabalho analisar a possível função desses genes nesse processo em células humanas não transformadas. Foi otimizado um sistema experimental para o estudo de possíveis perturbações na manutenção da inativação do cromossomo X, onde a re-expressão de genes submetidos a esse processo pode ser monitorada. Nesse sistema foram identificados dois genes, MAOA e GYG2, cujo padrão de expressão no X inativo difere do previamente descrito. Demonstrou-se que baixos níveis de expressão do gene XIST foram suficientes para manter seu RNA associado ao X inativo, conservando o estado silenciado desse cromossomo. Além disso, foram obtidos indicativos de que a inibição de XIST em fibroblastos humanos gera uma diminuição da viabilidade celular. Foi possível demonstrar que DNMT1 é necessária para a manutenção da metilação global do genoma em células humanas não transformadas, e que eXISTe um mecanismo de compensação da inibição desse gene que leva ao aumento da expressão de DNMT3B. Ainda se observou que a repressão de DNMT1 não é suficiente para levar à reativação de genes no cromossomo X inativo. Além disso, a desmetilação encontrada nos promotores de MAOA e XIST não foi suficiente para levar à expressão destes genes nos cromossomo X inativo e ativo, respectivamente. Estes resultados enfatizam a necessidade de se estudar os mecanismos moleculares da ICX em humanos utilizando sistemas experimentais adequados para a análise de herança epigenética. / X chromosome inactivation (XCI) is the phenomenon through which one of the X chromosomes in female mammals is silenced to achieve dosage compensation related to males. It involves the expression of XIST gene exclusively from the inactive X, and the association of its RNA in cis in this chromosome. This leads to a series of epigenetic modifications in the chromatin of the inactive X (Xi) that guarantee a stable maintenance of the transcriptional silence through all the mitoses in the organism. One of these epigenetic modifications is DNA methylation, achieved mainly by the maintenance DNA methylase DNMT1. The roles of XIST and DNMT1 in the maintenance phase of XCI are controversial in humans. Therefore, the main goal of this present work was to analyze some of the possible functions of these genes in this process in untransformed human cells. An experimental system was optimized to study possible disturbances in maintenance of XCI, where the re-expression of genes submitted to this process could be monitored. In this system we identified two genes, MAOA and GYG2, whose pattern of expression on the Xi, differed from what had been previously described. It was demonstrated that low levels of XIST expression were sufficient to keep its RNA associated to the Xi, assuring the silenced state of this chromosome. Besides, evidences have been found that XIST inhibition in human fibroblasts reduces cellular viability. It was possible to demonstrate that DNMT1 is necessary to the maintenance of global genome methylation in untransformed human cells, and the eXISTence of a compensation mechanism involving DNMT3B upregulation. It was also observed that repression of DNMT1 was not sufficient to reactivate genes of the Xi chromosome. Additionally, demethylation of MAOA and XIST promoters was not enough to cause expression of these genes on the inactive and active Xs, respectively. All these results emphasize the requirement of studying the molecular mechanisms of XCI in humans using experimental systems appropriate for the analysis of epigenetic inheritance.
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Single-Molecule and Super-Resolution Fluorescence Studies of the Structure and Function of Telomerase and TelomereWu, John Yanyun January 2012 (has links)
Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.
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Chemical biology studies on the structures and biological functions of nucleic acids / 核酸の構造と生物活性についてのケミカルバイオロジー研究Li, Yue 23 March 2016 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19527号 / 理博第4187号 / 新制||理||1601(附属図書館) / 32563 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 藤井 紀子 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM
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Padrão de Inativação do Cromossomo X e Expressão de microRNAs X-específicos na Pré-Eclâmpsia / X-chomosome Inactivation Pattern and of MicroRNAs X-specific Expression in PreeclampsiaOliveira, Adriane Araujo de 27 January 2010 (has links)
As síndromes hipertensivas gestacionais estão entre as maiores causas de morte materna e fetal. Entre elas destaca-se a pré-eclâmpsia (PE), que caracteriza-se pelo aumento da pressão arterial e proteinúria, a partir da 20ª semana de gestação. Embora sua etiologia seja ainda discutida, o papel dos fatores genéticos é amplamente aceito. Alterações do padrão da inativação do cromossomo X, processo epigenético encontrado em mamíferos com placenta, têm sido encontradas em algumas doenças que ocorrem exclusivamente em mulheres. O XIST é um gene chave nesse processo. Por outro lado, muitos microRNAs (pequenos RNAs não codificantes) são expressos abundantemente na placenta humana e alguns estão mapeados no cromossomo X. O objetivo do presente trabalho foi a verificação do padrão de inativação do cromossomo X e da expressão dos genes XIST e dos microRNAs X-específicos miR-221, miR-222 e mir-223 em mulheres com PE. O ensaio de HUMARA (receptor de andrógeno humano) utilizando PCR convencional e digestão com a enzima sensível à metilação HpaII foi analisado de forma qualitativa (visualização em gel de poliacrilamida) e semi-quantitativa (sequenciamento), sendo realizado a partir de sangue periférico (todas as amostras) e de tecido placentário [apenas das placentas de fetos femininos (17 amostras)]. Para o estudo do padrão de expressão foi obtido cDNA por transcrição reversa, a partir de RNA total extraído do tecido placentário (30 amostras).. A análise foi realizada por meio de PCR em tempo real. Foram utilizados os testes de Qui-quadrado e de t-Student, além do modelo linear generalizado para a análise estatística. Não houve diferença estatisticamente significativa para o parâmetro de inativação do cromossomo X entre os grupos controle e de PE, independente do tipo de tecido estudado (sangue ou placenta) quando foram aplicados os ensaios de HUMARA qualitativo e semi-quantitativo. Para o gene XIST e o microRNA miR-221 não foi evidenciada diferença estatisticamente significativa entre os grupos controle e de PE. O microRNA miR-223 não apresentou transcritos detectáveis em nenhum dos grupos de estudo. Para o microRNA miR-222, houve diferença estatisticamente significativa, sendo que no grupo de PE a expressão foi mais elevada. Não foi encontrada associação entre o padrão de inativação do cromossomo X e a expressão do gene XIST e dos microRNAs estudados. Embora a inativação preferencial do cromossomo X tenha sido encontrada nos dois grupos, padrões de inativação preferencial extrema foram verificados em um número maior de casos com PE. Os resultados mostram que o miR-222 apresenta potencial para ser utilizado como marcador molecular da PE, sugerindo também que exista uma diminuição na expressão de seus genes-alvo que devem ser estudados como candidatos na patogênese da doença. / The gestational hypertensive syndromes are among the major causes of maternal and fetal death. The Preeclampsia (PE) is the most prevalent of those syndromes and it is characterized by the increase of blood pressure and proteinury, which start from to 20th week of gestation. Although its ethiology is argued actually the genetics factors have been accepted. Alterations in pattern of chromosome X inactivation, epigenetic process of mammals with placenta have been found in some diseases that occur exclusively in women. XIST is a key gene in this process. On the other hand very microRNAs (small RNAs no coding) are overexpressed in human placenta and someones are located at X choromosome. The subject of this research was verify the patterns of chromosome X inactivation and the expression of the XIST gene and X-specific microRNAs in women affected by PE. In the HUMARA (human androgen receptor) assay was carried out with peripheral blood (all samples) and placental tissue [only female fetuses placentas (17 samples)]. The conventional PCR and digestion methodology with HpaII enzyme were employed and the result was analysed to qualitative (polyacrilamide gel) and semi quantitative (sequencing) form. The cDNA was obtained to gene expression study by reverse transcription reaction from placenta total RNA (30 samples). Gene expression assay was carried out with real time PCR. To statistical analyze was used the Qui-square and t-Student tests besides the widespread lineal model. There was not statistical significant differences to chormosome X inactivation parameter among the groups control and PE independently of the tissue studied (blood or placenta) when the qualitative and semi quantitative HUMARA assay were applied. To XIST and miR-221 were not evidenced significant statistical differences among the groups control and of PE. The miR-223 did not show detectable transcripts to any group studied. The miR-222 expression was more elevated in the PE group than control group and this difference was significant statistically. In the present study was not found association among the chromosome X inactivation pattern and the gene expression of XIST and the microRNAs studied. Although the chromosome X inactivation have been found in the two groups the preferential chromosome X inactivation patterns were verified in a great number of cases in PE group. The results showed that miR-222 has a potential to be employed as molecular marker of PE also suggesting the existence of a decrease in expression of its target genes which have to be investigated like candidates to disease pathogenesis.
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Analysis of the role of nuclear organization during random X chromosome inactivation / Analyse du role de l’organisation nucleaire dans l’inactivation du chromosome XPollex, Tim 19 September 2014 (has links)
Chez les mammifères femelles, le processus d’inactivation du chromosome X (XCI) assure la compensation de dose entre les deux sexes. Chez la souris, l’inactivation du X est établie de manière aléatoire dans l’épiblaste au stade blastocyste et peut être récapitulée in vitro dans les cellules souches embryonnaires. L’ARN non-codant Xist, exprimé à partir du centre d’inactivation du X (Xic), est le régulateur principal de ce processus. Il décore en cis le chromosome choisi pour être inactivé et initie la répression de ses gènes. Ainsi, de manière remarquable, les deux chromosomes X sont traités différemment pendant l’initiation de la XCI malgré leur homologie de séquence et leur localisation au sein d’un même noyau. De manière remarquable, ce processus implique le traitement différentiel de deux chromosomes homologues au sein d’un même noyau, avec des changements d’environnements nucléaires et chromatiniens entre le X actif et le X inactif. Il a été proposé que la localisation nucléaire pourrait jouer un rôle important dans l’initiation de l’expression monoallélique des gènes, non seulement pour l’initiation de la XCI mais aussi pour des processus tels que l’exclusion allélique dans les cellules lymphoides. Par exemple, l’association de loci avec des compartiments hétérochromatiques nucléaires et l’association en trans de loci homologues pourraient être impliquées dans la régulation des gènes monoalléliques. Ainsi, j’ai utilisé le système bactérien TetR/TetO afin d’analyser le rôle de l’organisation nucléaire du chromosome X et du Xic dans l’initiation de la XCI. J’ai pu montrer que si le recrutement des protéines de fusion TetR-LaminB1 ou -Cbx5 au niveau de la cassette TetO insérée dans le Xic permet la répression des gènes à proximité, cet évènement n’est pas toujours accompagné d’une relocalisation nucléaire. De plus, l’association forcée du Xic avec la périphérie nucléaire (TetR-LaminB1) n’a pas d’influence sur le choix du chromosome X à inactiver. Enfin, si la relocalisation des deux Xic à la périphérie nucléaire induit une réduction des évènements d’association en trans entre les deux loci, elle n’a pas d’effet sur l’initiation de l’inactivation. En résumé, ces résultats suggèrent que l’organisation nucléaire du chromosome X et du Xic et l’association des Xic en trans ne sont pas des facteurs déterministes pour le choix et l’initiation de la XCI, mais pourraient être le résultat des changements d’expression des gènes liés à l’X au cours de la différenciation des cellules souches ainsi que de l’augmentation de l’expression de Xist.Enfin, j’ai également analysé le rôle de la protéine CTCF, qui a été proposée pour être importante dans l’organisation structurale du génome, dans le contexte du Xic et de l’initiation de l’inactivation. Ainsi, le recrutement de CTCF au niveau de cassette TetO insérée dans le Xic induit localement une réduction mineure des interactions en cis et la répression des gènes du Xic, à l’exception de Xist dont l’expression est augmentée. Pour autant, la présence ectopique de CTCF n’a pas d’incidence majeure sur l’organisation générale du Xic. / X-chromosome inactivation (XCI) ensures dosage compensation in female mammals. Random XCI is established in the epiblast of female mouse embryos and can be recapitulated in vitro in differentiating embryonic stem cells (ESCs). The major regulator of XCI is the long non-coding RNA Xist, which is expressed from the X-inactivation center (Xic), covers the chromosome in cis and initiates gene silencing. During XCI, the two X chromosomes are treated very differently, despite their homology and the fact that they reside in the same nucleus. Nuclear localization has been hypothesized to play a role in monoallelic gene regulation, not only during XCI but also in other contexts. For example, association with heterochromatin and homologous trans interactions (“pairing”) have been implicated in the establishment of monoallelic gene expression in lymphoid cells and transient pairing has been suggested to participate in symmetry breaking during random XCI. Using the bacterial tetO/tetR system to alter the subnuclear localization and environment of one or both Xics, we have tested the function of subnuclear localization and trans interactions between the Xic loci during initiation of XCI. Using stable expression and reversible binding of TetR fusion proteins (e.g. LaminB1, Cbx5) we show that binding of these proteins can induce local gene repression and chromatin changes, although this is not always associated with subnuclear relocalization. We further show that the forced association of the Xic with the nuclear envelope, does not impact on the choice-making process during XCI. In particular, tethering both Xics to the nuclear lamina during early ESC differentiation resulted in a substantial reduction of homologous pairing events, but had no obvious impact on the onset of random, monoallelic Xist expression. Taken together, our results suggest that nuclear localization and trans interactions of the Xic may be downstream events rather than causal in the regulation of the XCI process.Furthermore, we recruited CTCF, a protein suggested to be involved in structural organization of the genome, to the Xic using the tetO/tetR system. Upon binding of CTCF the overall structure of the Xic remained unaltered though few cis interactions appeared to be weakened, which was accompanied by gene repression in the Xic. Surprisingly, the only upregulated gene in the Xic was Xist in ESCs and during differentiation, which demonstrates that the induced minor changes of cis interactions might impact on gene regulation in the Xic.
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Padrão de Inativação do Cromossomo X e Expressão de microRNAs X-específicos na Pré-Eclâmpsia / X-chomosome Inactivation Pattern and of MicroRNAs X-specific Expression in PreeclampsiaAdriane Araujo de Oliveira 27 January 2010 (has links)
As síndromes hipertensivas gestacionais estão entre as maiores causas de morte materna e fetal. Entre elas destaca-se a pré-eclâmpsia (PE), que caracteriza-se pelo aumento da pressão arterial e proteinúria, a partir da 20ª semana de gestação. Embora sua etiologia seja ainda discutida, o papel dos fatores genéticos é amplamente aceito. Alterações do padrão da inativação do cromossomo X, processo epigenético encontrado em mamíferos com placenta, têm sido encontradas em algumas doenças que ocorrem exclusivamente em mulheres. O XIST é um gene chave nesse processo. Por outro lado, muitos microRNAs (pequenos RNAs não codificantes) são expressos abundantemente na placenta humana e alguns estão mapeados no cromossomo X. O objetivo do presente trabalho foi a verificação do padrão de inativação do cromossomo X e da expressão dos genes XIST e dos microRNAs X-específicos miR-221, miR-222 e mir-223 em mulheres com PE. O ensaio de HUMARA (receptor de andrógeno humano) utilizando PCR convencional e digestão com a enzima sensível à metilação HpaII foi analisado de forma qualitativa (visualização em gel de poliacrilamida) e semi-quantitativa (sequenciamento), sendo realizado a partir de sangue periférico (todas as amostras) e de tecido placentário [apenas das placentas de fetos femininos (17 amostras)]. Para o estudo do padrão de expressão foi obtido cDNA por transcrição reversa, a partir de RNA total extraído do tecido placentário (30 amostras).. A análise foi realizada por meio de PCR em tempo real. Foram utilizados os testes de Qui-quadrado e de t-Student, além do modelo linear generalizado para a análise estatística. Não houve diferença estatisticamente significativa para o parâmetro de inativação do cromossomo X entre os grupos controle e de PE, independente do tipo de tecido estudado (sangue ou placenta) quando foram aplicados os ensaios de HUMARA qualitativo e semi-quantitativo. Para o gene XIST e o microRNA miR-221 não foi evidenciada diferença estatisticamente significativa entre os grupos controle e de PE. O microRNA miR-223 não apresentou transcritos detectáveis em nenhum dos grupos de estudo. Para o microRNA miR-222, houve diferença estatisticamente significativa, sendo que no grupo de PE a expressão foi mais elevada. Não foi encontrada associação entre o padrão de inativação do cromossomo X e a expressão do gene XIST e dos microRNAs estudados. Embora a inativação preferencial do cromossomo X tenha sido encontrada nos dois grupos, padrões de inativação preferencial extrema foram verificados em um número maior de casos com PE. Os resultados mostram que o miR-222 apresenta potencial para ser utilizado como marcador molecular da PE, sugerindo também que exista uma diminuição na expressão de seus genes-alvo que devem ser estudados como candidatos na patogênese da doença. / The gestational hypertensive syndromes are among the major causes of maternal and fetal death. The Preeclampsia (PE) is the most prevalent of those syndromes and it is characterized by the increase of blood pressure and proteinury, which start from to 20th week of gestation. Although its ethiology is argued actually the genetics factors have been accepted. Alterations in pattern of chromosome X inactivation, epigenetic process of mammals with placenta have been found in some diseases that occur exclusively in women. XIST is a key gene in this process. On the other hand very microRNAs (small RNAs no coding) are overexpressed in human placenta and someones are located at X choromosome. The subject of this research was verify the patterns of chromosome X inactivation and the expression of the XIST gene and X-specific microRNAs in women affected by PE. In the HUMARA (human androgen receptor) assay was carried out with peripheral blood (all samples) and placental tissue [only female fetuses placentas (17 samples)]. The conventional PCR and digestion methodology with HpaII enzyme were employed and the result was analysed to qualitative (polyacrilamide gel) and semi quantitative (sequencing) form. The cDNA was obtained to gene expression study by reverse transcription reaction from placenta total RNA (30 samples). Gene expression assay was carried out with real time PCR. To statistical analyze was used the Qui-square and t-Student tests besides the widespread lineal model. There was not statistical significant differences to chormosome X inactivation parameter among the groups control and PE independently of the tissue studied (blood or placenta) when the qualitative and semi quantitative HUMARA assay were applied. To XIST and miR-221 were not evidenced significant statistical differences among the groups control and of PE. The miR-223 did not show detectable transcripts to any group studied. The miR-222 expression was more elevated in the PE group than control group and this difference was significant statistically. In the present study was not found association among the chromosome X inactivation pattern and the gene expression of XIST and the microRNAs studied. Although the chromosome X inactivation have been found in the two groups the preferential chromosome X inactivation patterns were verified in a great number of cases in PE group. The results showed that miR-222 has a potential to be employed as molecular marker of PE also suggesting the existence of a decrease in expression of its target genes which have to be investigated like candidates to disease pathogenesis.
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